Vibrio cholerae， the causative pathogen of the life-threatening infection cholera， encodes two copies of β-ketoacyl-acyl carrier protein synthase III (vcFabH1 and vcFabH2). vcFabH1 and vcFabH2 are pathogenic proteins associated with fatty acid synthesis， lipid metabolism， and potential applications in biofuel production. Our biochemical assays characterize vcFabH1 as exhibiting specificity for acetyl-CoA and CoA thioesters with short acyl chains， similar to that observed for FabH homologs found in most gram-negative bacteria. vcFabH2 prefers medium chain-length acyl-CoA thioesters， particularly octanoyl-CoA， which is a pattern of specificity rarely seen in bacteria. Structural characterization of one vcFabH1 and six vcFabH2 structures determined in either apo form or in complex with acetyl-CoA/octanoyl-CoA indicate that the substrate-binding pockets of vcFabH1 and vcFabH2 are of different sizes， accounting for variations in substrate chain-length specificity. An unusual and unique feature of vcFabH2 is its C-terminal fragment that interacts with both the substrate-entrance loop and the dimer interface of the enzyme. Our discovery of the pattern of substrate specificity of both vcFabH1 and vcFabH2 can potentially aid the development of novel antibacterial agents against V. cholerae. Additionally， the distinctive substrate preference of FabH2 in V. cholerae and related facultative anaerobes conceivably make it an attractive component of genetically engineered bacteria used for commercial biofuel production.